Fluorescent compounds (fluorophores) have been widely used in immunoassays, flow cytometry, fluorescence microscopy, and DNA sequencing. To date, the sensitivity of such assays has been limited by the spectral properties of available fluorophores.
In particular, automated DNA sequencing has become an important tool in molecular biology. The most successful strategies utilize the Sanger dideoxy chain termination method with either a 5'-fluorophore-labeled primer or fluorophore-labeled dideoxynucleotide triphosphates to generate a series of fragments. The resultant fragments are separated by electrophoresis. Careful selection of the enzyme, fluorophore, and reaction conditions has increased the size of DNA fragments that can be sequenced by such techniques from a hundred to nearly a thousand bases. For example, Applied Biosystems Incorporated (ABI) reports the ability to sequence nearly a 700 base pair stretch of DNA within 13 hours using a fluorophore-labeled primer. Despite advances in automated sequencing, the current technology does not allow single-run sequencing of kilobase and greater lengths of DNA. This limit is imposed, in part, by fluorophore detection and resolution. Signal detection could be improved by the use of fluorophores with more ideal spectral properties.
Recently, DNA sequencing systems have been described based on the use of a novel set of four chain-terminating nucleotides, each carrying a different chemically tuned succinylfluorescein dye distinguished by its fluorescent emission. Prober, J. M., et al., Science 238:336-341, 1987; European Patent Application No. 87305848.1.
The effect of peripheral substitution of fluoro and cyano groups on the electronic properties of silicon dihydroxy phthalocyanine has been modelled. Hale, P. D., et al., J. Am. Chem. Soc. 109(20):5943-5947, 1987. The calculated wavelength of absorbance for the parent silicon phthalocyanine was predicted to be 673 nm while the octacyano- and octafluoro- derivatives had calculated transitions at 685 and 756 nm, respectively. No mention of fluorescence is made in the report.
Introduction of phenoxy and thiophenoxy substituents into the phthalocyanine macrocycle reportedly led to an appreciable red shift in the long wavelength band in the visible absorbance spectra. Derkacheva, V. M., and E. A. Luk'yanets, J. Gen. Chem. USSR 50:1874-1878, 1980. The sulfur substituted phthalocyanines were said to be more red-shifted in absorbance than the oxygen substituted derivatives and, in either case, the 3-substituted phthalocyanines were reportedly more greatly shifted than the 4-isomers. No fluorescence data was reported. Of the compounds discussed in the Luk'yanets report, only the metal free derivatives are potential fluorophores. The cobalt and copper analogs are nonfluorescent. Metal free phthalocyanines are not capable of being rendered reactive or water soluble by the techniques described herein since the metal free specie is unstable to some of these techniques, such as chlorosulfonation.
The application of aluminum phthalocyanines to simultaneous, multicomponent fluorescence analysis such as in nucleic acid sequence analysis, flow cytometry, immuno- or nucleic acid probe assays requires the preparation of a family of tetherable, water-soluble derivatives with a common excitation wavelength and yet different emission wavelengths, with maximal spectral resolution between each family member. For DNA sequence analysis, four such fluorophores are desired.
Ideal fluorophores have five characteristics: a readily accessible excitation wavelength with a large molar absorptivity, a high fluorescence quantum yield, a large Stokes shift (&gt;50 nm), emission at long wavelengths (greater than 600 nm), and a sharp emission profile (full width at half maximum, FWHM&lt;40 nm).
Aluminum phthalocyanine (AlPc) has nearly ideal spectral properties. Excitation of aluminum phthalocyanine at 350 nm results in emission at 685 nm with fluorescence quantum yield (.phi..sub.f) of 0.58. Brannon, J. H., and D. Magde, J. Amer. Chem. Soc., 102(1):62-65, 1980. Aluminum phthalocyanine (AlPc)is composed of a highly conjugated macrocycle and a trivalent aluminum atom. The structure of the parent AlPc fluorophore is shown below. L is a ligand such as OH when the AlPc is in water. The trivalent aluminum atom provides axial ligation which serves to reduce aggregation and thereby increases fluorescence in solution. ##STR2##
In a related application, U.S. Ser. No. 366,971, filed Jun. 14, 1989, the present inventors have disclosed water-soluble phthalocyanine compounds that are monomerically conjugated to biochemical moieties.
In therapeutic applications, aluminum phthalocyanine sulfonates have been determined to be effective in directed cell killing. Ben-Hur, E. and I. Rosenthal, Photochem. Photobiol. 42(2):129-133, 1985. The advantage that phthalocyanines have over other photodynamic agents is their large molar absorptivity in the red region of the visible spectrum. The large molar extinction coefficient coupled with the transparency of tissue at these red wavelengths provides for more efficient light penetration and subsequently more effective treatment of subcutaneous malignancies. Pursuant to the present invention, aluminum phthalocyanine derivatives red-shifted from the parent compound will provide an even greater depth of penetration and enable even more effective treatments. Derivatives attached to biological moieties such as probes or antibodies can be targeted to specific cell populations.
Closely related to the phthalocyanines are the tetrabenztriazaporphyrins, referred to herein as TBTAPs. Barrett, P. A., et al, J. Chem. See. 1809-1828, 1937. The only structural difference is the replacement of the nitrogen at position twenty of the phthalocyanine with a substituted carbon. No substituted derivatives of these compounds have been reported to date. Nor have any tetherable or water soluble analogs been reported. The spectral and luminescent properties of magnesium and palladium benzoporphyrins have been reported. Solovev, K. N. et al., Opt. Spectrosc. 27:24-29, 1969. Neither aluminum, substituted, tetherable or water-soluble derivatives are discussed.